HDFSC were compared to human bone marrow mesenchymal stem cells (hBMSC) and differentiated primary human gingival fibroblasts (hGiF), as well as permanent gingival carcinoma cells (Ca9-22).In patients with periodontitis, it is highly likely that local (progenitor) cells encounter pathogenic bacteria. The purpose of this Porphyromonas gingivalis  and Fusobacterium nucleatum (F. nucleatum). The viability of the cells and pathogens under anaerobic conditions, as well as interactions in terms of adherence and internalization, were examined. Additionally, the release of pro-inflammatory interleukin-8 (IL-8) and anti-inflammatory interleukin-10 (IL-10) was quantified via enzyme-linked immunosorbent assay. The bacteria adhered less efficiently to hDFSC compared to Ca9-22 . Similar results were observed for host cell internalization . Statistically significantly less IL-8 was secreted from hDFSC after stimulation with F. nucleatum and P. gingivalis in comparison with hGiF . The IL-10 response of the differentiated cells was found to be low in relation to their pro-inflammatory IL-8 response.The different cell types were investigated in a co-culture system with The results indicate that dental stem cells are less prone to interactions with pathogenic bacteria than differentiated cells in an anaerobic environment. Moreover, during bacterial challenge, the stem cell immune response seems to be more towards an anti-inflammatory reaction. For a potential future therapeutic use of hDFSC, these findings support the idea of a save application. To determine the biological role of dental stem cells in their natural environment and to examine the save potential therapeutic application for tissue regeneration, the understanding of cell–bacteria interactions is of great importance The periodontal tissues are composed of a variety of different cell types. Recently, stem cells have been reported to be part of this cellular environment The dental follicle harbors human dental follicle stem cells (hDFSC), and is present in impacted teeth, which are commonly extracted and disposed of medical waste in dental practice The periodontal pocket is predisposed towards interdependencies of local cells and putative pathogens, which have been one focus of periodontal research F. nucleatum and P. gingivalis can frequently be found in diseased periodontal tissues and they play important roles in the initiation and progression of periodontitis P. gingivalis is able to induce strong cytokine and chemokine expression in gingival epithelial and other host cells, which has been shown to positively correlate with the adhesive/invasive potential of the infecting strain F. nucleatum and P. gingivalis are obligate anaerobic bacteria that can only exist in areas of the oral cavity with low oxygen partial pressure . However, functional interaction studies of hDFSC and oral pathogenic bacteria in their natural anaerobic atmosphere are lacking.The pathogenic bacteria Thus, the focus of our study was to investigate the interactions of hDFSC with periodontal pathogens. The results were compared with cells at various stages of differentiation: human bone marrow mesenchymal stem cells (hBMSC), human gingival fibroblasts (hGiF), and the permanent gingival carcinoma cell line Ca9-22. We hypothesized that hDFSC might show a different immunological response than differentiated cells after co-culture with periodontal pathogens because of their anti-inflammatory and tissue regenerative function in the injured or inflamed tissue The stem cells were obtained from donors undergoing surgical treatment at either the Department of Cardiac Surgery or the Department of Oral Surgery at the University of Rostock. This study conforms to the Declaration of Helsinki, and all cell donors gave their informed written consent. The experimental protocol and further experiments were reviewed and approved by the Ethics Committee of the University of Rostock (No. A 2011 119 and No. A 2011 91).2, 37°C).Ca9-22 was provided by the Japanese Collection of Research Bioresources Cell Bank Osaka, Japan. The cells were cultured in DMEM supplemented with 10% FCS in cell culture flasks  . Isolation and culturing of hBMSC was performed as described by Gaebel et al.et al.HGiF were cultured in DMEM supplemented with 10% FSC , F. nucleatum ATCC 23726, and ATCC 25586  were purchased from commercial providers. The bacteria were grown in PYG medium supplemented with 5 µg/ml hemin and 1% vitamin K in an anaerobic atmosphere .Bacteria were tested for their survival in cell culture medium prior to the co-culture experiments. They were grown in PYG medium supplemented with 5 µg/ml hemin and 1% vitamin K to the stationary phase. Subsequently, bacteria were centrifuged, washed in PBS, and each bacterial suspension was diluted in DMEM 1∶10. The optical density (OD) was measured at 600 nm from timepoint 0 every hour to timepoint 12 h with a final measurement after 24 h.th to 6th passage were harvested and seeded at a density of 8×103 cells/well in a 24-well culture plate, and maintained in 2 ml of medium. Subsequently, cells were grown in an anaerobic workstation  at 37°C for 24, 48, and 76 h, and numbers of viable cells were counted in a Neubauer hemacytometer after trypan blue staining.Cells were tested for their tolerance towards oxygen-free conditions. Cell-cultures at the 43–1×104 cells/well in a 24-well culture plate, and maintained in 1 ml medium. For each experiment, the final concentration of the bacterial suspension was determined by measurement of the optical density at 600 nm (OD600) to obtain 1×108 cells/ml, and dilutions were made to achieve the desired MOI. The bacterial inoculum was confirmed by counting of the colony-forming units (CFU). After reaching confluence, the cells were infected with live bacteria with a multiplicity of infection (MOI) of 1∶100 and incubated at 37°C in an anaerobic atmosphere.For co-culture, hDFSC, hBMSC, hGiF, and Ca9-22 were seeded at a density of 8×10For fluorescence microscopy  the samples were stained with BacLight Live/Dead . The staining was documented with an attached digital camera .3 cells/ml) were cultured in DMEM and grown to a monolayer. The bacteria were grown in PYG medium at 37°C under an anaerobic atmosphere. The bacterial density was adjusted to MOI 1∶100 in DMEM and added to the cell monolayer. After 2 h, the cells were washed and subsequently detached by adding 200 µl 0.25% trypsin/0.5 mM EDTA for 5 min. To quantify bound bacteria, the cells were lysed with distilled water and the number of bacteria in the lysate was assessed by viable counts. For quantification of internalized bacteria, the monolayers were washed with PBS. Medium containing gentamicin (300 mg/ml) and metronidazole (200 mg/ml) was added after 2 h and incubated for another 2 h. After 4 h, the same procedure was performed to quantify the internalized bacteria. The number of viable bacteria was confirmed by counting CFUs on agar plates incubated under an anaerobic atmosphere at 37°C.The eukaryotic cells , sputter-coated with gold, and examined with an electron microscope .For scanning electron microscopy (SEM), coverslips with hDFSC co-cultures were fixed in a 2.5% glutardialdehyde solution. They were subsequently washed with 0.1 M sodium acetate buffer (pH 7.3) and dehydrated in a graded series of ethanol. Finally, coverslips were subjected to critical-point drying with COIL-8 and IL-10 concentrations were measured in the supernatant of the bacteria–cell co-culture after 1, 2, 4, and 24 h of incubation by enzyme-linked immunosorbent assay (ELISA), using a commercially available kit  according to the manufacturer's instructions.All results are presented as means ± standard deviation (SD). Statistical analyses were carried out by T-test . Differences were considered statistically significant at *p<0.05, **p<0.01. All experiments were performed with at least four biological replicates (n = 4).et al. The establishment of the anaerobic co-culture system as described by Kriebel The bacterial growth was determined in cell culture medium under anaerobic conditions. All four tested species showed normal bacterial growth . From thP. gingivalis W50, W83, F. nucleatum ATTC 23726, or ATTC 25586, employing a MOI of 1∶100. Attachment to and invasion of hDFSC by oral pathogenic bacteria was measured, and was compared with the results obtained with hBMSC, hGiF, and Ca9-22. All bacterial species were able to adhere to and invade the hDFSC. Notably, in all co-cultures, Ca9-22 cells showed the highest susceptibility to bacterial adhesion, which was statistically significant for F. nucleatum ATCC 2326 and P. gingivalis W50. Roughly the same number of bacteria adhered to the fibroblasts compared to the hDFSC. In contrast, in all co-cultures, the hBMSC were statistically significantly less prone to bacterial adhesion. SEM confirmed bacterial attachment to the hDFSC  for 1, 2, 4, and 24 h, IL-8 and IL-10 was detected in the supernatant of all four cell lines by ELISA (After co-culture with either live by ELISA  and S3. F. nucleatum ATCC 255886 infection). Overall, F. nucleatum was the most efficient stimulatory bacterium for the secondary cytokine response. The strongest IL-8 response was noted after 24 h for hGiF co-cultured with F.nucleatum ATCC 23726, reaching 1964 pg/ml.Of note, there were statistically significantly lower IL-8 secretion levels in hDFSC and hBMSC supernatants compared with hGiF and Ca9-22. At all timepoints, hGiF consistently showed the highest IL-8 response  was higher than the initial IL-8 response measured for all cells. After 4 h of co-culture, the IL-8 response from hGiF and Ca9-22 increased significantly, whereas the hDFSC and hBMSC remained at almost the same level of IL-10 secretion, but showed only a small increase in IL-8 response.in vitro, since in bacterially infected periodontal tissues, a mutual influence of local (progenitor) cells and anaerobic bacteria is highly likely in vitro co-culture experiments with cells and anaerobic bacteria is the limited lifespan of these microorganisms under an aerobic atmosphere The purpose of the present study was to comparatively characterize interactions between dental stem cell and periodontal pathogens et al.P. gingivalis into gingival epithelial cells. It is known that P. gingivalis can recognize different host cell types and is capable of targeting different specific and distinct eukaryotic signaling pathways to induce uptake into the cell et al.F. nucleatum is able to adhere to and invade gingival epithelial cells.Host cell invasion is regarded as an important tool of pathogenic bacteria to afford protection from the host immune system, and this invasion contributes to tissue damage et al. P. gingivalis attachment for the two tested cell lines in that study . It is not clear what kind of receptors were responsible, but these authors were able to show a saturation plateau for each cell line when all bacteria were bound. Whether PRRs (pathogen recognition receptors) play a role in this context still needs to be determined.In this study, we reveal the differential susceptibility of dental stem cells compared with hBMSC, hGiF, and Ca9-22 towards the very important bacterial pathogenicity traits of host cell adherence and internalization. A significantly lower bacterial adherence and internalization capacity was observed in dental stem cells compared with hGiF and the epithelial cell line Ca9-22. Variations in the cell-surface structure, as well as differences in the process of invasion, depending on the cell line and its level of differentiation might explain the decreased bacterial attachment and internalization found in hDFSC and hBMSC compared with hGiF or Ca9-22 co-cultures. Furthermore, the expression of varying amounts of specific cell-surface receptors that allow the attachment of pathogens may contribute to the different results for the adherence. Huard-Delcourt in vitro pathogen growth et al.et al.Moreover, a novel stem cell property has been described, which possibly contributes to a reduced bacterial adherence and internalization. BMSCs have recently been shown to impede F. nucleatum and P. gingivalis, the cytokine release was assessed. IL-8, an important signaling molecule in periodontitis In vitro, IL-8 secretion is triggered by invading bacteria, particularly F. nucleatum; similar observations for P. gingivalis have been made, with different cell lines et al.To define differences in the immunological reactions of the cells after co-culture with P. gingivalis and F. nucleatum with hDFSC, the IL-8 secretion was significantly less stimulated compared with hGiF and Ca9-22 cells. All four cell lines under investigation showed the highest IL-8 production after co-culture with F. nucleatum ATCC 23726. The decrease in IL-8 levels after 24 h in the P. gingivalis W50 co-culture is most likely due to the activity of cytokine degrading enzymes of this species Our results show that after the co-culture of F. nucleatum, while P. gingivalis LPS signals via TLR2 P.gingivalis and gingipains also activate TLR4 The differences in the cytokine response between the cells might be rooted in the specificity of the innate immune system that is based on a broad spectrum of PRRs that recognize highly conserved pathogen-associated molecular patterns (PAMPs) in vitro findings of this work underline the potential immuno-modulating character of local dental progenitor cells in the context of bacterially induced inflammatory processes.However, recent reports suggest that human dental mesenchymal stem cells, as well as hBMSC, possess immuno-regulating and anti-inflammatory properties In vitro, we compared hDFSC, hBMSC, differentiated primary gingival fibroblasts, and the permanent tumor cell line Ca9-22 in an anaerobic environment. The results of this study indicate that there are differences between cell lines at various stages of differentiation concerning the tolerance towards bacterial infections. For future potential dental stem cell therapies, the findings presented here might be of clinical relevance.We demonstrate that hDFSC (and hBMSC) show less interaction with periodontal pathogens concerning adherence and internalization. Moreover, in this experimental setup, the cytokine secretion of these cells favored an anti-inflammatory reaction. However, better anaerobic survival of the stem cells could lead to a better chance of tissue regeneration after infection and damage, which seems to be reasonable from an evolutionary perspective. With respect to dental tissues, it can be concluded that hDFSC show a similar behavior to hBMSC in this study.Figure S1Scanning electron microscope images of F. nucleatum ATCC 23727 attachment to hDFSC.(TIF)Click here for additional data file.Figure S2Scanning electron microscope images of F. nucleatum ATCC 23727 attachment to hDFSC.(TIF)Click here for additional data file.Table S1Adherence and internalization. The attachment and invasion levels are expressed as the percentage of live bacteria retrieved following cell lysis relative to the total number of bacteria at each timepoint. The assays were carried out as described in the text. Attachment and invasion levels of hDFSC and hBMSC were compared with Ca9-22 cells. Values represent the means ± SD, *p<0.05, **p = 0.01 (T-test), n = 4.(XLSX)Click here for additional data file.Table S2IL-8 secretion. IL-8 levels were assayed by ELISA. Absorbance was read at 450 nm. Values represent the means ± SD, *p<0.05, **p = 0.01 (T-test), n = 4.(XLSX)Click here for additional data file.Table S3Il-10 secretion. IL-8 levels were assayed by ELISA. Absorbance was read at 450 nm. Values represent the means ± SD, *p<0.05, **p = 0.01 (T-test), n = 4.(XLSX)Click here for additional data file.


Chronic stress is a risk factor for the development of psychiatric disorders, some of which involve dysfunction of the prefrontal cortex (PFC). There is a higher prevalence of these chronic stress-related psychiatric disorders during adolescence, when the PFC has not yet fully matured. In the present work we studied the effect of repeated stress during adolescence on synaptic function in the PFC in adolescence and adulthood. To this end, adolescent Sprague-Dawley rats were subjected to seven consecutive days of restraint stress. Afterward, both synaptic transmission and short- and long-term synaptic plasticity were evaluated in layer 1 of medial-PFC (mPFC) slices from adolescent and adult rats. We found that repeated stress significantly reduced the amplitude of evoked field excitatory post-synaptic potential (fEPSP) in the mPFC. Isolation of excitatory transmission reveled that lower-amplitude fEPSPs were associated with a reduction in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated transmission. We also found that repeated stress significantly decreased long-term depression (LTD). Interestingly, AMPA/kainate receptor-mediated transmission and LTD were recovered in adult animals that experienced a three-week stress-free recovery period. The data indicates that the changes in synaptic transmission and plasticity in the mPFC induced by repeated stress during adolescence are reversed in adulthood after a stress-free period. Stress is a biological response that allows adaptation to environmental threats  and water, and at a room temperature of (21 ± 1°C). All animals were handled and weighed daily after weaning. All procedures related to animal experimentation were in accordance with NIH guidelines and were approved by the Institutional Animal Ethics Committee of the Universidad de Valparaíso, Chile. Efforts were made to minimize the number of animals used and their suffering.Male Sprague-Dawley rats were housed in groups of 3–4 per home cage under a 12 h light / dark cycle (lights at 8:00 am), n = 8) or stress (n = 8) group. Control animals were housed in a separate room and not subjected to any type of stress. Stress group animals were subjected to restraint stress in their home cages for 3 h per day for seven consecutive days (from PND 42–49) in a cylindrical acrylic restrainer (8 cm in diameter × 22 cm long). To evaluate synaptic function at adolescence and adulthood, four animals per group were subjected to electrophysiological experiments 1 day  or 21 days  after the end of the stress protocol. The percentage of body weight gain (net change in weight in grams × 100/weight at the beginning of the stress protocol n = 8 per group) and adrenal weight  were measured during the stress protocol to monitor physiological stress response  35–55  was positioned in layer 1 of the prelimbic cortex, the dorsal part of the mPFC  antagonist. To isolate the AMPA/kainate receptor-mediated fEPSP, the slices were incubated in normal ACSF containing 50 μMD-2-amino-5-phosphonovaleric acid (APV), a N-Methyl-D-aspartate (NMDA) receptor (NMDAR) antagonist, and 1 μM PTX. To isolate the NMDAR-mediated fEPSP, the slices were incubated in ACSF without Mg2+ containing 5 μM 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), an AMPA/kainate antagonist, and 1 μM PTX.To isolate the excitatory component of the fEPSPs, the slices were incubated in normal ACSF containing 1 μM picrotoxin (PTX), a gamma-aminobutyric acid (GABA)-type A receptor  = 365.4; p < 0.001; n = 8 per group). However, repeated application of the restraint protocol significantly reduced body weight gain  = 401.6; p < 0.0001; interaction between the passage of days and stress protocol: F = 15.05; p < 0.0001). We also found that seven days of restraint significantly increased adrenal weight . These results indicate that seven days of restraint was effective in activating a chronic stress response.Figure We first characterized synaptic transmission in layer 1 of the mPFC of non-stressed animals. Figure F = 0.022; p = 0.881), suggesting that there were no significant inhibitory transmissions in the evoked fEPSPs. However, the application of PTX resulted in a negative field potential following the fEPSP  = 0.061; p = 0.804). We then isolated the NMDAR-mediated fEPSPs by superfusing AMPA/kainate inhibitor CNQX and PTX in ACSF lacking Mg2+. Treatment with both agents resulted in the almost complete abolition of post-synaptic response. However, strong stimulation (50 μA) evoked a field potential of lower amplitude than that evoked in ACSF , the fEPSP evoked by the second pulse had a lower amplitude than that evoked by the first pulse, with PPR values <1.0. This indicates that synapses of layer 1 have paired-pulse depression. To evaluate the contribution of Ca2+ to short-term synaptic plasticity, we tested PPS at a low Ca2+ concentration (0.5 mM) and observed that the amplitude of the fEPSP evoked by the second pulse was higher than that evoked by the first pulse. This resulted in PPR values >1.0, indicative of paired-pulse facilitation . Superfusing APV into the bath produced a significant difference in the fEPSP amplitude compared to LFS condition, in both early-phase  and late-phase LTD  = 21.39, p < 0.001). Bonferroni post hoc comparison revealed that the fEPSP amplitudes of stressed animals were significantly lower than those of the controls at stimulus intensities of 30, 40 (p < 0.01) and 50 μA (p < 0.001). In contrast, we did not find significant differences between adult control and stressed rats  = 1.43; p = 0.231). We found no differences between groups in the amplitude of the fiber volley in either adolescence  = 0.156; p = 0.693) or adulthood  = 1.184; p = 0.278; Figures We next evaluated the effect of repeated stress during adolescence on synaptic transmission in the mPFC in adolescence and adulthood. We measured fEPSP amplitude evoked by different intensities of electrical stimulation and found as shown previously  = 1.392; p = 0.239) and adult animals  = 2.36; p = 0.125) at different ISIs. This suggests that the reduction in synaptic transmission in the mPFC induced by repeated stress was not related to alterations in the probability of neurotransmitter release.Given that the reduction of synaptic transmission is attributed to decreased neurotransmitter release in the presynaptic domain  = 26.26; p < 0.0001). Bonferroni post hoc comparison showed significantly lower fEPSP amplitudes in the stressed group, measured in adolescence at 30, 40 and 50 μA, than in the control  = 0.065; p = 0.798). Altogether, the data suggest that the decrease in synaptic function in the mPFC induced by repeated stress was independent of inhibitory transmission, and that excitatory transmission was affected.The reduction of synaptic transmission in the mPFC could also be attributed to increased inhibitory transmission. To examine this possibility, we blocked inhibitory transmission by applying PTX, a GABAl Figure . We analF = 27.14; p < 0.0001). The Bonferroni post hoc comparison showed significantly lower AMPA/kainate mediated-receptor fEPSP amplitude in the stressed group at 30, 40 and 50 μA of stimulus intensity  = 0.092; p = 0.761). Subsequently, we evaluated the effect of repeated stress in NMDAR-mediated transmission. As shown in Figures F = 1.859; p = 0.175) and adult  = 1.18; p = 0.28) control and stressed group animals. These findings suggest that repeated restraint stress decreases AMPA/kainate receptor-mediated glutamatergic transmission in layer 1 of the mPFC, which returns to control levels in adulthood after stress-free period.To evaluate the contribution of AMPA/kainate and NMDA receptors to repeated stress-induced impairment in synaptic transmission we pharmacologically isolated both NMDAR- and AMPA/kainate receptor-mediated transmissions. As shown in Figure y Figure . The redF = 13.59; p < 0.001; Figures p < 0.01; Bonferroni post hoc comparison) but not of early LTD .We investigated the effect of repeated stress on LTD induction (early LTD) and expression (late LTD) in adolescence and adulthood  = 0.474; p = 0.497) on the magnitude of LTD. We found no significant differences between groups in the magnitude of either early  or late LTD (control = 74.1 ± 6.5%; stress = 71.8 ± 9.1%; p > 0.05; Figure We then evaluated the effect of repeated stress on LTD in adult rats. Figure in vivo models, where dopaminergic fibers are preserved, LTP is commonly evoked in the mPFC, and blocking D1 receptor antagonists blocks LTP (Gurden et al., We found that synaptic activity in layer 1 of the mPFC was mostly mediated by AMPA/kainate and NMDA receptors, which also displayed paired-pulse depression and NMDA-dependent LTD. However, under our working conditions, LTP was not induced. Similar results have been published previously related to the glutamatergic receptors involved in synaptic response (Hirsch and Crepel, The fEPSP recorded in layer 1 in our study may in part reflect the activation of deeper synapses in the same neuronal population due to collaterals from layer 1 contacting pyramidal cells in deeper regions. However, previous data have shown that horizontal fibers that cross layer 1 almost exclusively contact monosynaptically the apical tufts of pyramidal neurons of layers 2/3 and 5, with little activation of synapses in deeper layers (Cauller and Connors, The apical dendritic tufts in the mPFC are innervated by horizontal fibers that arrive mainly from the mediodorsal thalamus, the amygdala and distal cortical areas (Bacon et al., A or NMDA receptors. It has been reported that chronic stress, as well as corticosterone administration, decreases both AMPA/kainate receptor-mediated transmission and the number of AMPA/kainate receptors in the mPFC by proteasome-mediated degradation (Yuen et al., We found that 7 days of restraint stress during adolescence decreased fEPSP amplitude in layer 1 of the mPFC. This reduction is not attributed to differences in the amount of stimulated afferent fibers or changes in probability of neurotransmitter release, because we did not find differences in either the amplitude of the presynaptic volley or the magnitude of the PPR, respectively. Our results indicate that the repeated stress-induced decrease in basal synaptic transmission during adolescence is mediated by post-synaptic alterations in AMPA/kainate dependent transmission, but not because of alterations in GABAWe also found that repeated stress during adolescence decreased the expression of LTD, but not its induction. Given that the induction of LTD in the mPFC seems to be dependent on NMDAR Figure , this reWe found that both basal synaptic transmission and LTD were recovered in adulthood after a stress-free period. Interestingly, the recovery of synaptic transmission and LTD was accompanied by a recovery of AMPA/kainate receptor-mediated transmission. Earlier studies showed that chronic stress-induced dendritic atrophy in the mPFC during adolescence was reversed in adulthood (Radley et al., What processes are involved in the observed effects? The evidence suggests that stress response is more intense during adolescence (Pruessner et al., We previously found that repeated stress during adolescence impairs the recall of the extinction of conditioned fear, an impairment that was reversed in adulthood (Negrón-Oyarzo et al., Adolescence is often described as a developmental window of vulnerability to psychiatric disorders (Paus et al., IN-O designed research, performed the experiments and analyzed the results. AD-S and PMC supervised the performance of the experiments and data analysis. IN-O and PMC wrote the paper and all authors critically revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Phaseolus vulgaris leucoagglutinin (PHA-L) into the BLA indicated 25.63% increase in the number of PHA-L-immunopositive terminal-like structures in the ventral subiculum (v-Sub) of epileptic mice as compared to control mice. These data suggest that the projections from the basal nucleus at BLA to the vSub in epileptic mice are resistant to epilepsy-induced damage. Consequently, these epileptic mice exhibit partially impairment but not total loss of context-dependent fear memory. Epileptic mice also show increased c-Fos expression in the BLA and vSub when subjected to contextual memory test, suggesting the participation of these two brain areas in foot shock-dependent fear conditioning. These results indicate the presence of functional neural connections between the BLA-vSub regions that participate in learning and memory in epileptic mice.In this study, we investigated the reorganized basolateral amygdala (BLA)-subiculum pathway in a status epilepticus (SE) mouse model with epileptic episodes induced by pilocarpine. We have previously observed a dramatic loss of neurons in the CA1–3 fields of the hippocampus in epileptic mice. Herein, we observed a 43–57% reduction in the number of neurons in the BLA of epileptic mice. However, injection of an anterograde tracer,  The amygdala, hippocampus, parahippocampal regions and their interconnected neural circuits have been implicated in the fear memory consolidation processes  for several hours showed histopathological alterations throughout the limbic system Coulter, . The affLesions in the basolateral amygdala (BLA) region abolished conditional freezing in response to contextual cues associated with foot shocks, suggesting that BLA is important in mediating conditioned fear responses  into the BLA.Handling and care of all animals were in compliance with the NIH guidelines for animal research. All animal experiments were approved by the Institutional Animal Care and Use Committee at the Tan Tock Seng Hospital/National Neuroscience Institute. Male Swiss mice (25–30 g) used for all experiments in this study were treated as described previously ; (2) SE2m with PHA-L (n = 11); (3) control with CTB (n = 9); and (4) SE2m with CTB (n = 10). Four control mice (2 injected with PHA-L and 2 injected with CTB) and 7 SE2m mice (four injected with PHA-L and three injected with CTB) were excluded from data analysis due to incorrect injection sites.A total 50 mice were used . Twenty-one of thirty-three pilocarpine-injected mice survived  at two months after injection (SE2m). These 17 saline-injected and 21 pilocarpine-injected mice were randomly allocated to four experimental groups: (1) control with PHA-L  in distilled water or 2.5% PHA-L  in 0.1 M phosphate buffered saline (pH 7.4) in a glass micropipette with a diameter of 20–30 μm was delivered iontophoretically into the BLA using a positive current of 5 μA  over 10 min. Animals were sacrificed at 7 days after CTB or PHA-L injection and were perfused transcardially using 50 ml of saline, 100 ml of 4% paraformaldehyde and 0.15% picric acid in 0.1 M PB at pH 7.4 for 30 min. Coronal brain sections of 40 μm thickness were cut using a cryostat and the serial brain sections were collected in individual wells of 24-well plates.2O2 and 0.05% DAB  in TBS for 20 min, mounted on glass slides, covered with coverslips, and photographed using an image capture system. PHA-L stained sections were also counterstained with cresyl fast violet (CFV).For immunohistochemistry, free-floating brain sections were first washed with TBST  containing 0.1% Triton-X 100). Brain sections were then incubated overnight at 4°C in mouse anti-NeuN antibody , goat anti-c-Fos antibody , goat anti-PHA-L antibody , or goat anti-CTB antibody , then washed three times with TBST before incubation in their respective secondary antibodies diluted 1:500 with TBST . Brain sections were then incubated in avidin-biotin complex (ABC) reagent in TBST for 2 h, washed with TBS and allowed to react in a solution of 0.12% HDouble labeling of PHA-L with calbindin (CB), calretinin (CR) or parvalbumin (PV) was carried out by incubating brain sections in goat anti-PHA-L (1:1000), together with rabbit anti-CB (1:2000), anti-CR (1:1500) or anti-PV (1:1500) for 48 h at 4°C. The brain sections were washed and incubated in biotinylated horse anti-goat IgG (1:500) and swine anti-rabbit IgG (1:100) for 4 h at room temperature, then incubated in ABC solution for 2 h at room temperature before reaction with 3,3′-diaminobenzidine (DAB)-Nickel solution for 20 min. Subsequently, rabbit peroxidase anti-peroxidase  solution was added to the brain sections and left overnight to incubate, and sections were then developed with DAB alone.2) normalized with cell loss/area of SE animals as mean value ± standard deviation (SD).Every third sections were stained with antibodies against NeuN or c-Fos for cell counting. BLA was determined anteroposterior (AP) to Bregma . In the BLA, the total number of neurons per area in the specified regions of interest (ROI) in control mice and epileptic (SE2m) mice were quantified and the data were normalized with area shrinkage of SE animals in the specific brain regions. The c-Fos-expressing neurons in specific brain regions such as the hippocampus , the dorsal (dSub) and ventral (vSub) subiculum , were also counted and presented as density  at specific ROI were counted and the data were normalized with area shrinkage of SE animals. ROI is defined as the specific brain region of interest  based on their anatomical locations in the brain. Images of the ROI were taken at the focal planes of the ROI that showed clearest and in-focused structures. The numbers of CB, CR or PV-immunopositive neurons showing physical contacts with the PHA-L-labeled terminal-like structures were also counted as described above. In the vSub, CTB labeled neurons were counted and presented as density (No./mm2) normalized with the area in SE animals relative to the control animals. Imaging-Pro Plus  was used for all quantitative analysis in this study. The neuronal profile counts were blinded. Statistical analysis were done between the control and the SE2m mice at different time points using one-way ANOVA followed by Student-Newman-Keuls post hoc tests (for experiments with more than two groups of data). For comparing experiments with two groups of data, an independent samples test (t-test) was used instead. A p-value of less than 0.05 is considered statistically significant.PHA-L-immunopositive terminal-like structures in the vSub were visualized at high (400×) magnification (10× eyepiece magnification plus 40× objective lens) using a Zeiss wide field light microscope. These terminal-like structures were identified based on their visibly enlarged structures seen on PHA-L-immunopositive fibers. The numbers of these enlarged structures on fibers per mmFear conditioning and retention memory tests were conducted using a Y-maze with stainless-steel grid floor (30 × 10.5 × 14.5 cm) as described previously , status epilepticus/context  group, control/context with foot shock group  and status epilepticus/context with foot shock group . Each mouse was handled for 1 min per day for 3 days before the start of training to reduce stress in the later training and test. The arms were thoroughly cleaned with 10% ethanol solution directly after each training and testing session. On Day 1 (habituation day), mice were allowed to explore Y-maze freely for 8 min. The total numbers of entrance to each arm were recorded. On Day 2 (training day), mice in N/CS and SE/CS groups were placed in the shock arm that was blocked off from the rest of the maze. After 120 s, mice in the above two groups received four foot shocks each . Mice were returned to the home cages 1 min after the last foot shock. The total freezing times of each animal after each foot shock were recorded. The freezing time for each session was presented as a percentage of total observation. Mice in N/C and SE/C groups were placed in the shock arm for the same period of time, without receiving foot shocks. On Day 3 (test day), mice were placed in a non-shock arm and allowed to access all arms of the maze for 4 min. The total freezing time and latency to enter shock or another non-shock arm were recorded. The percentage of the number of the shock arm entries to all arm entries was calculated. No shock was delivered on Day 3.For behavioral experiments, a total of 42 mice were used . Twenty out of the thirty pilocarpine-injected mice survived  at 2 months after injection (SE2m). Four experiment groups were designed for behavioral test that included control/context group  and 0.1514 ± 0.0198, 0.2396 ± 0.032 and 0.1894 ± 0.0153 mm2 in the control mice respectively. There were no significant differences in the La and B nuclei areas  between the epileptic and control mice. However, a significant decreased in the AB nucleus area  was observed in the epileptic mice as compared to the control mice. Therefore, to account for this atrophy in the epileptic mice, total neurons per area in the epileptic mice were normalized to the same area of the control mice. NeuN immunocytochemistry showed 47.15, 43.26 and 56.80% reduction in total number of neurons at the La, B and AB nuclei of the amygdala, respectively, in the epileptic mice (SE2m) as compared to the control mice  at the La , B  and AB  nuclei of the amygdala  in the vSub of epileptic mice (0.7233 ± 0.0617 mm2) as compared to control mice (0.6200 ± 0.0584 mm2). NeuN immunocytochemistry showed a significant 28.06% reduction in total number of neurons at the vSub in the epileptic mice as compared to the control mice  at the vSub in the epileptic mice  compared to that of control mice .PHA-L was iontophoretically injected into the BLA in control or epileptic mice and these mice were observed for PHA-L labeling in the vSub Figures . Images t-test, ***p < 0.001, **p < 0.01).To determine the contact points of PHA-L-immunopositive en passant and terminal-like structures in vSub, we carried out double immunostaining of PHA-L with CB, CR or PV. We found visible contacts between PHA-L immunopositive en passant and terminal-like structures and CB, CR or PV-immunopositive neurons in vSub of mice in both the control Figures  and epilt-test, p > 0.05).Next, we iontophoretically injected CTB into the BLA of control and epileptic mice to determine the retrograde connections from BLA. CTB-immunopositive neurons were found mainly in the ipsilateral vSub in both the control Figure  and the post hoc multiple comparisons and a paired t-test indicated that the N/CS group exhibited significantly longer freezing times after each of the four foot shocks as compared to the SE/CS, SE/C and N/C groups , but SE/CS group showed significantly longer freezing times than N/C and SE/C groups after each of the four shocks .We next investigated if the structural changes in epileptic mice were associated with functional consequences. Since epilepsy has been shown to affect hippocampal functions, we selected Y-maze contextual fear memory test to assess learning and memory function in the control and epileptic mice. During habituation (Day 1), mice in all groups explored all three arms of the maze evenly and displayed no freezing. There were no differences in the percentage of shock arm entries Figure , Day 1 opost hoc test, ***p < 0.001, Figure post hoc test, ***p < 0.001, Figure post hoc test, **p < 0.01) and the SE/CS group compared to the SE/C group  and SE/CS group than SE/C  as compared to the N/C, SE/C and SE/CS groups .c-Fos is required for hippocampal functions  after the fear memory test at Day 3  as compared to those in N/C .The density of c-Fos-immunopositive neurons in N/CS group was increased significantly in the dorsal (dSub; Figures  Figures , SE/C (F Figures  and SE/C Figures . However Figures  were obsPilocarpine-induced SE, when not interrupted by drug administration, is able to induce reorganization and anatomical damage in brain regions such as the olfactory cortex, amygdala, thalamus, hippocampus and neocortex (Curia et al., Neuronal loss and gliosis in the amygdala have been reported in a large number of neuropathological studies using animal TLE models and in patients with TLE (Turski et al., c-Fos is an immediate early gene. It is part of the activator protein-1 transcription factor and has been postulated to participate in the molecular mechanisms of learning and memory. c-Fos was selected as a marker of neuronal activity because it has been used to assess region-specific activity during fear conditioning in previous studies (Campeau et al., The shock-trained epileptic mice in our study entered the shock arm with shorter latencies and at higher frequencies as compared to the shock-trained control. This indicates that fear memory of epileptic mice was significantly impaired. However, we still observed increased c-Fos expression in the dentate gyrus of epileptic mice. This increase in amygdala c-Fos expression and that epileptic mice with significant neuronal loss in the hippocampus could still demonstrate freezing responses upon entering the shock arm suggest that another neural pathway is involved in contextual fear memory. The relatively undamaged BLA in the epileptic mice could potentially still play a role in fear memory even when the hippocampus is substantially damaged. Moreover, increased c-Fos expression, particularly in the BLA of control mice upon contextual fear memory test provided further evidence on the involvement of the BLA in foot shock dependent-fear memory. These results are in agreement with previous reports showing that cues-associated foot shock increases c-Fos mRNA and protein expression in the amygdala (Campeau et al., Here, we report that epileptic mice have significantly higher numbers of CR, CB or PV-immunopositive cells contacted by PHA-L labeled en passant and terminal-like structures in the v-Sub as compared to control mice. These connections are important for synaptic excitability and plasticity in the efferent and afferent pathways of BLA. The presence of ~50% more terminal-like structures in the v-Sub of epileptic mice that were labeled with PHA-L injected at BLA may provide the neuroanatomical basis to support the enhancement of interaction between BLA and v-Sub in epilepsy. These findings suggest an enhanced or compensatory higher excitability of neurons in the amygdala of epileptic mice.Our data suggest that an increase in aberrant network connections and compensatory mechanisms in the parahippocampal formation may contribute to neurological deficits in the epileptic mice and possibly, also in humans with TLE. Therefore, a panoramic analysis beyond the temporal lobe of these epileptic mice is essential for better understanding the lesions and compensatory reorganization of circuitry in epilepsy. Currently, reorganizations of the neural circuits in several brain regions of this mouse epilepsy model including the hippocampus (Ma et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Further to these in vitro studies, we carried out in vivo experiments to verify hemodynamic effects of BK analogs exhibiting variable susceptibility toward vascular and blood plasma peptidases. Rats were anesthetized and instrumented to record blood pressure and heart rate responses to bolus intravenous (i.v.) injection of increasing doses of BK, B-9972 , BK-Arg, BK-His-Leu or BK-Ala-Pro, in the absence or presence of specific inhibitors. In some experiments, pulsed Doppler flow probes measured hindquarter Doppler shift in response to i.v. injections of kinins. BK caused rapid, transient and dose-related hypotensive effects. These effects were potentiated ∼15-fold by the angiotensin converting enzyme (ACE) inhibitor, enalaprilat, but extensively inhibited by icatibant (a B2R antagonist) and not influenced by the Arg-carboxypeptidase (CP) inhibitor (Plummer’s inhibitor). The hypotensive responses elicited by the peptidase-resistant B2R agonist, B-9972, were not affected by enalaprilat, but were inhibited by icatibant. The hypotensive responses to BK-Arg were abolished by pre-treatment with either the Arg-CP inhibitor or icatibant, pharmacologically evidencing BK regeneration. The hypotensive effects of BK-His-Leu and BK-Ala-Pro, previously reported as ACE-activated substrates, were abolished by icatibant, but not by enalaprilat. In vivo regeneration of BK from these two C-terminally extended analogs with no affinity for the B2R must follow alternative cleavage rules involving unidentified carboxypeptidase(s) when ACE is blocked. The transient hypotensive responses to BK and three tested analogs coincided with concomitant vasodilation . Together, these results provide in vivo evidence that interesting hypotensive and vasodilator effects can be extracted from prodrug peptides that behave as peptidase-activated B2R agonists.We reported evidence of bradykinin (BK) regeneration from C-terminal extended BK sequences that behave as peptidase-activated B The B2R is constitutively expressed in many tissues, and its activation is believed to play a major role in the cardioprotective effects of BK during hypertension and other clinical and experimental conditions, such as cardiac failure, ischemia, myocardial infarction, and pulmonary hypertension  toward resident vascular peptidases. The BK-related peptides tested and their hypothetical metabolism are presented in Figure  The natural BK sequence was compared to the peptidase-resistant agonist, B-9972, and to C-terminally prolonged BK homologs designed to retain little affinity for the B2R (Table 1), but that are presumably activated by vascular or blood plasma peptidases, such as ACE  or arginine carboxypeptidases (BK-Arg).Although there have been very few attempts to use BK or a derivative in pharmacotherapy, the cardiovascular benefits of Bddressed . Neverthddressed , reductiddressed . All thets of BK . Inspireagonists . In contAll experimental methods and animal care procedures were reviewed and approved by the Animal Care and Handling Committee of Laval University, in accordance with the Canadian Council on Animal Care. Experiments were performed on male Sprague-Dawley rats (300–375 g) purchased from Charles River Laboratories . The rats were housed two per cage in a temperature-controlled room (22 ± 1°C) on a 12:12-h light-dark cycle (lights on at 0600). Animals had free access to normal chow diet and tap water. They were allowed to acclimate to their environmental conditions for 1 week prior to being studied.3H]enalaprilat binding to the enzyme expressed in cells (summarized in Table 1), except for BK-Arg. To compare the affinity of BK-Arg to that of BK for ACE, HEK 293a cells that transiently expressed human recombinant ACE C-terminally fused with the mCherry fluorescent protein were used to perform a [3H]enalaprilat binding assay as described  and had two separate catheters implanted; one into the right jugular vein [for intravenous (i.v.) injection] and the other into the left femoral artery (for direct measurement of blood pressure and heart rate (HR). In some experiments, a pulsed Doppler flow probe  coupled to a computer-based data acquisition system  to continuously record pulsate arterial pressure. The animals were maintained under anesthesia and were warmed with a heated pad. The mean arterial pressure and HR were simultaneously calculated by the Acknowledge software (version 3.9.1) for Windows  and displayed continuously.9-BK. Peptides were dissolved in isotonic saline (0.9% NaCl) containing 0.1% BSA to prevent the adsorption of peptide to the glassware and plastic surfaces. All i.v. injections were given as 100 μl boluses which were washed in with a further 100 μl of saline (the dead space of the catheter). Only one peptide was tested per group of rats and each injection started with saline-BSA 0.1% followed by the lowest dose of peptide. The next dose was administered once all recorded cardiovascular parameters had returned to baseline after the previous injection . At the end of the experiments each animal was euthanized with an overdose of sodium pentobarbitone .The experiments were initiated after allowing at least 20 min for stabilization. Then, baseline measurements of HR and phasic and mean arterial blood pressure (MAP) were made over a period of 10 min. A dose response curve was then obtained by recording changes in blood pressure and HR elicited by i.v. injection of peptide vehicle followed by increasing doses  of one of these peptides: BK, BK-His-Leu, B-9972, BK-Arg, BK-Ala-Pro or des-Arg2R antagonist, icatibant (Hoe 140)  . B-9858  is a selective B1R antagonist  following a 10 min period of baseline measurements of HR and blood pressure. Fifteen minutes later, dose-response curves to BK, BK-His-Leu, B-9972, BK-Arg, or BK-Ala-Pro were obtained in the indicated group of rats, as described above. Further experiments were made in rats pretreated with the Plummer’s inhibitor (mercaptomethyl- 3-guanidinoethylthiopropanoic acid), a high affinity inhibitor of arginine carboxypeptidases that is an arginine analog . In thesdykinin) . In thesdykinin) , with detagonist  that hasnist , BK (700 ng/kg), B-9972 (150 ng/kg), BK-Arg (2 μg/kg), and BK-His-Leu (400 ng/kg) in random order. For each agent, the dose interpolated from the dose-response curves produces a sizeable hypotensive effect of 15–30 mmHg. All i.v. injections were given as 100 μl boluses which were washed in with a further 100 μl of saline. The next dose was administered once all recorded cardiovascular parameters had returned to baseline after the previous injection . At the end of the experiments the rats were euthanized with an overdose of anesthetic .9-BK were purchased from Bachem , the B2R antagonist icatibant, from Phoenix Pharmaceuticals , enalaprilat dehydrate, from Kemprotec Ltd.  and the Plummer’s carboxypeptidase inhibitor, from Calbiochem . B-9972  is a peptidase-resistant agonist of the B2R  followed by the Dunnett’s test (repeated comparison with a common control). The effects of peptidase inhibitors on the hypotensive responses to BK and alternate kinin receptor agonists were assessed by using ANOVA followed by the Dunnett’s test. A value of P < 0.05 was considered significant.Data are presented as means ± SEM. Radioligand binding data were fitted by non-linear regression to a one-site competition equation using a least-square method  and ICTable  While no significant changes in basal values of MAP were noted between the enalaprilat and Plummer’s inhibitor pretreated groups, a slight but significant reduction in basal MAP was found between the icatibant pretreated group and the control group. Furthermore, slight but significant reductions in basal HR were noted between the icatibant and Plummer’s inhibitor pretreated groups and the control group, while a small significant increase in basal HR was found between the enalaprilat pretreated groups and the control group.Baseline values for MAP and HR measured in the untreated control group or 15 min after i.v. pretreatment with enalaprilat, icatibant or the Plummer’s inhibitor are shown in Figures 2A,B, the i.v. injection of increasing doses of BK in anesthetized rats caused a rapid and transient dose-related decrease in MAP; responses were significantly greater than the saline-BSA vehicle for BK doses superior or equal to 0.4 μg/kg . Tachycardia, (preceded by a significant bradycardia only at the dose of 6.4 μg/kg), accompanied the hypotensive responses only at 0.4 μg/kg and above . In rats pretreated with enalaprilat, the hypotensive effect of BK was potentiated ∼15-fold ; the duration of the hypotensive episodes were not different from those of controls . Pretreatment with icatibant significantly and extensively inhibited the dose-dependent hypotensive response to BK in the 0.4–6.4 μg/kg dose range , while pretreatment with the Plummer’s inhibitor had no effect , consistent with a minor role of Arg-carboxypeptidases in the metabolism of BK. The B1R agonist des-Arg9-BK had no significant effect on MAP at the dose of 0.025 μg/kg, while slight but significant decreases in MAP were noted at 0.1 μg/kg and above  when compared to vehicle values . The comparatively small effects of the fragment des-Arg9-BK exclude that responses to BK are mediated by the B1Rs via metabolism by Arg-carboxypeptidases. Accordingly, the B1R antagonist B-9858, did not significantly modify the effect of BK .As expected and shown in 2R agonist B-9972 decreased MAP in a dose-dependent manner . The hypotensive response elicited by B-9972 was significant at doses of 0.1 to 6.4 μg/kg when compared to vehicle values . The hypotensive episodes induced by B-9972 had the same temporal profile than those elicited by BK  except for the highest dose of the analog, that was associated with a half-recovery time significantly longer than that of a similar dose of BK . Consistent with its low affinity for ACE (Table 1) and constrained C-terminal structure, the dose-dependent hypotensive response to B-9972 was not affected by pretreatment with enalaprilat. However, consistent with its direct agonist action on the B2R, the effects of the 0.1–1.6 μg/kg doses of B-9972 were significantly abated by pretreatment with icatibant; thus this competitive antagonist reduced the apparent potency of B-9972 by about 10-fold . Biphasic effects of B-9972 on the HR were generally comparable to that of BK but of higher amplitudes at 1.6–6.4 μg/kg .The i.v. injection of increasing doses of the peptidase-resistant BFigures 5A,B, the i.v. injection of increasing doses of BK-Arg elicited significant and dose-dependent decreases in MAP. The reductions in MAP were significant at doses of 0.4 to 6.4 μg/kg  when compared to vehicle values. This peptide is predicted to be an indirect activator of the B2R, via its conversion to BK . Consistently, its in vivo effects were abolished in rats pre-treated with either the Plummer’s carboxypeptidase inhibitor or icatibant . BK regeneration from BK-Arg should not involve the carboxydipeptidase ACE . While micromolar concentration levels of BK displace the radioligand from the peptidase, as previously reported, BK-Arg at 10 μM had little effect in this competition assay . Whereas pretreatment with enalaprilat did not affect the hypotensive response to 0.025 to 1.6 μg/kg BK-Arg, the pretreatment was found to significantly potentiate the hypotensive response elicited by the highest dose of BK-Arg . This might result from the potentiation of regenerated BK, considering that BK-Arg has a low intrinsic affinity for ACE . Tachycardia accompanied the hypotensive responses at doses of 0.1 to 6.4 μg/kg .As shown in dase ACE . To veriFigures 6A,B shows that BK-His-Leu (0.025–6.4 μg/kg) decreased MAP in a significant and dose-dependent manner. The reductions in MAP were significant at doses of 0.4 to 6.4 μg/kg  when compared to vehicle values. BK-His-Leu is predicted to be an indirect activator of the B2R, via its conversion to BK, which should involve the carboxydipeptidase ACE . Surprisingly, the dose-dependent hypotensive response to BK-His-Leu, which has very little affinity for the B2R but a good one for ACE (Table 1), was not significantly affected by pretreatment with enalaprilat. However, the hypotensive effect of BK-His-Leu was abolished by pretreatment with icatibant , raising the possibility that in the presence of enalaprilat, BK regeneration from BK-His-Leu might involve unidentified peptidase(s) via alternate cleavage rules. Tachycardia accompanied the hypotensive responses at doses of 1.6 and 6.4 μg/kg .Figures 5A and 6A); however, this was variable and statistics on recovery half-time for hypotensive episodes generally failed to show statistical difference from those recorded following similar doses of BK.Both pro-drug peptides BK-Arg and BK-His-Leu had a tendency to produce prolonged hypotensive episodes with, occasionally, a slow onset . Figure 7 shows that BK-Ala-Pro decreased MAP in a dose-dependent manner . While icatibant abated the effects of BK-Ala-Pro , enalaprilat pretreatment unexpectedly potentiated this peptide .BK-Ala-Pro is an alternate ACE substrate that may regenerate BK following the action of ACE  signal and HR induced by intravenous injections of vehicle, BK and alternate kinin receptor agonists administered at doses producing a sizeable hypotensive effect of 15–30 mmHg. Thus, we found that, for each tested agonist, the observed hypotensive response coincided with a concomitant increase in the mean Doppler shift signal and tachycardia when compared with vehicle values. The increase in mHDS might reflect a change in vascular resistance (vasodilation) redistributing flow to the hindlimb muscles, as previously shown in conscious rats , suggesting actions mediated along a continuum of physiological effects mediated by cardiovascular B2Rs.ous rats . The amp2Rs using a variety of ligand design strategies that mainly exploit their susceptibility toward vascular peptidases. The vascular and plasmatic localization of ACE and kininase I–type carboxypeptidases makes them ideally situated to regulate BK activity on B2Rs of vascular tissue, thus avoiding extravascular stimulation of these receptors .Aberrant protease signaling pathways have been implicated in several diseases, ranging from cardiovascular disorders to cancer, making them attractive therapeutic targets in drug development. To this end, the development of protease resistant drugs might be very useful in order to prolong beneficial effects of endogenous substances otherwise too rapidly degraded. Protease-activated prodrugs can also be successfully exploited to improve drug delivery to areas where protease expression is higher than in normal tissues, and might contribute to reduce off-target side effects. Although the protective role of kinins in the circulation is increasingly recognized, there have been very few attempts to use BK or a derivative in cardiovascular therapeutics. In the present study, we exploited the distribution of ectopeptidases expressed in the vasculature and blood plasma to examine in anesthetized rats the feasibility of extracting hemodynamic effects of the stimulation of endothelial B2R agonist that is also an effective ACE substrate (Table 1). As expected, the rapid and transient hypotensive response to systemic administration of BK was greatly enhanced by pretreatment with an ACE inhibitor, but extensively inhibited in the presence of a B2R antagonist, and remained unchanged in the presence of a specific inhibitor of arginine carboxypeptidases . These results further confirm the important role played by the ACE, as the main BK-inactivating peptidase in the extracellular space , the i.v. injection of B-9972 caused hypotensive responses that were not affected by pretreatment with enalaprilat, but significantly reduced by pretreatment with icatibant. Further, despite its diminished affinity for the B2R relative to BK, as judged from a binding assay to recombinant B2R conducted at 0°C in the presence of peptidase inhibitors (Table 1), the analog B-9972 is a more potent hypotensive agent than BK in vivo ; B-9972 is nearly as potent as BK tested in the presence of enalaprilat. Although we might have expected that the synthetic peptide B-9972 exhibits prolonged hypotensive effects (independently of potency) as inferred from previous studies involving infusion of B-9972 or comparably designed peptides resistant to peptidases .We then tested the effects of the stable Broteases , 2009. Ises . Using the C-terminally extended peptide, BK-Arg, as a potential substrate of kininase I–type carboxypeptidases, we provided pharmacological evidence that BK-Arg behave as an Arg-carboxypeptidase-activated B2R agonist, as the hypotensive response to this analog was strongly inhibited by pretreatment with icatibant and the Plummer’s inhibitor, a mercapto analog of Arg . These results further support previous findings from this laboratory showing a loss of BK-Arg contractile potency in a venous contractile bioassay in the presence of the Plummer’s inhibitor (Table 1), it is thus plausible that the enhanced response was consecutive to a potentiation of regenerated BK.Extending a recent boratory , we provg of Arg  . In turn, regenerated BK may be considerably potentiated by ACE blockade, leading to such paradoxical results as a gain of apparent potency of BK-Ala-Pro in response to enalaprilat pretreatment . Therefore, BK-His-Leu and BK-Ala-Pro are certainly pro-drugs that release BK, but in a more complex metabolic context than that anticipated from previous in vitro experiments.Pharmacological evidence of B the B2R , was obtTable 1; ], the si1R to the transient hypotensive response elicited by the three C-terminally extented analogs appears unlikely as these peptides were shown to have little or no direct affinity for the B1R , in which the expression of inducible B1Rs should be minimal . However, this was variable and statistics on half-recovery time for hypotensive episodes generally failed to show statistical difference from those recorded following similar doses of BK under the applied protocol (bolus administration of peptides).Both pro-drug peptides BK-Arg and BK-His-Leu had a tendency to produce prolonged hypotensive episodes with, occasionally, a slow onset, which might result from a slow and progressive regeneration of BK following limited proteolysis , we demonstrate that the transient hypotensive response to BK and all tested alternate kinin receptor agonists coincided with a concomitant increase in the mHDS signal and tachycardia. The increase in mean Doppler shift might reflect a change in vascular resistance (vasodilation) redistributing flow to the hindlimb muscles, as previously shown by one of us in conscious rats  of the two general strategies, administration of a pro-peptide that releases BK vs. the use of a peptidase-resistant agonist like B-9972.The present study constitutes an MJ performed the experiments, analyzed the data, and reviewed drafts of the paper. LG provided unique reagents, analyzed the data, and reviewed drafts of the paper. XC-M performed some experiments, designed some of the experiments and reviewed drafts of the paper. HB and FM conceived and designed the experiments, analyzed the data, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper. All authors approved the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The antigenic characteristics of the Cap protein potentially may allow the development of a detection kit that could be applied to control PiCV infection. However, poor expression and poor protein solubility have hampered the production of recombinant Cap protein in the bacteria. This study was undertaken to develop the optimal expression of recombinant full-length Cap protein of PiCV using an E. coli expression system.Pigeon circovirus (PiCV) is considered to be a viral agent central to the development of young pigeon disease syndrome (YPDS). The Cap protein, a structural protein encoded by the cap gene was cloned and fused with different fusion partners including a His-tag, a GST-tag (glutathioine-S-transferase tag) and a Trx-His-tag (thioredoxin-His tag). The resulting constructs were then expressed after transformation into a number of different E. coli strains; these then had their protein expression evaluated. The expression of the recombinant Cap protein in E. coli was significantly increased when Cap protein was fused with either a GST-tag or a Trx-His tag rather than a His-tag. After various rare amino acid codons presented in the Cap protein were optimized to give the sequence rCapopt, the expression level of the GST-rCapopt in E. coli BL21(DE3) was further increased to a significant degree. The highest protein expression level of GST-rCapopt obtained was 394.27 ± 26.1 mg/L per liter using the E. coli strain BL21(DE3)-pLysS. Moreover, approximately 74.5% of the expressed GST-rCapopt was in soluble form, which is higher than the soluble Trx-His-rCapopt expressed using the BL21(DE3)-pLysS strain. After purification using a GST affinity column combined with ion-exchange chromatography, the purified recombinant GST-rCapopt protein was found to have good antigenic activity when tested against PiCV-infected pigeon sera.The PiCV E. coli-expressed full-length PiCV Cap protein has great potential in terms of large-scaled production and this should allow in the future the development of a serodiagnostic kit that is able to clinically detect PiCV infection in pigeons.These findings shows that the  Moreover, in above system, most of the expressed recombinant Cap protein was found to be in the form of inclusion bodies [E. coli expression system is still easier to carry out and is more cost-effective when applied to viral antigen production than the baculovirus-insect cell system, although the E. coli system does have some limitations.Pigeon circovirus (PiCV), is a non-enveloped virus and is considered to be the viral agent central to the development of young pigeon disease syndrome (YPDS). YPDS syndrome is a multifactorial disease that includes various unspecific clinical signs such as poor racing performance, weight loss, lethargy, anorexia, respiratory distress and diarrhea . At presdization -10. Enzyn system ,12. Howen bodies . The infE. coli expression system need to be overcome; these include making sure that the full-length of the Cap protein is expressed in E. coli and using an expression system in which the majority of Cap protein is produced in a soluble form rather than as inclusion bodies. If successful, this would not only allow the efficient purification of capsid protein on a scale that would allow an investigation of PiCV structural biology but also the purified recombinant protein would be potentially useful when developing diagnostic kits for the clinical detection of PiCV infection.To develop the Cap protein as coating antigen of an ELISA system, the above mentioned limitations associated with using an cap gene was fused to a series of different fusion tags in order to improve recombinant Cap (rCap) protein expression. The rCap was then expressed attached to three different expression tags in order to evaluate rCap fusion protein expression and production across a number of different E. coli strains. Three expression vectors were used, one harboring a glutathione-S-transferase (GST) tag, another harboring a 6xHis tag and finally, a third harboring a thioredoxin-6xHis (Trx-His); these were investigated to explore the effect of these very different fusion tags on the expression of rCap protein across various E. coli strains. In addition, optimizations of codon usage for various amino acids within the Cap gene were also carried out to give the rCapopt sequence and then the effect of these changes on expression of rCapopt in the various E. coli strains was assessed. Finally, purified rCapopt protein was examined in order to determine its antigenicity and therefore its usefulness in further serodiagnostic applications. To the best of our knowledge, the yield of E. coli expressed full-length rCapopt in this study after codon optimization of the cap gene is the highest known to date.In this study, the PiCV E. coli strains had a range of effects on the expression level of the two recombinant PiCV capsid proteins in E. coli allowing optimization of the protein purification protocol. To investigate the expression of PiCV capsid protein (Cap) using the prokaryotic expression system, the cap gene sequence was individually fused with three different tag sequences, His-tag (6xHis), glutathione-s-transferase tag (GST) and thioredoxin-His (Trx-His) in three distinct expression vectors  harboring pHis-Cap, pGST-Cap and pTrx-His-Cap were examined, there was no significant amount of rCap fusion protein present in the whole cell lysates after IPTG induction for 4 hrs -pLysS and BL21(DE3)-RIPL and their protein expression levels compared to that of E. coli BL21(DE3). The expression patterns of the rCap fusion protein in E. coli BL21(DE3)-pLysS and in E. coli BL21(DE3)-RIPL containing pHis-Cap, pGST-Cap, and pTrx-His-Cap are shown in Figures E. coli and, moreover, the E. coli strain used also plays a crucial role in expression and seems to affect the further application of these strains in large-scale production.As a result of the above findings, expression of the PiCV rCap fusion protein in E. coil when a Trx-His tag or GST tag on the N-terminus of rCap protein was used. The best expression was obtained using the strain BL21(DE3)-RIPL, which harbors extra copies of tRNAargU, proL, ileY, leuW. This is commercial E. coli host strain used for the gene expression of recombinant proteins that contain E. coli’s rare codons. The expression of Trx-His-rCap protein in strain BL21(DE3)-RIPL showing an expression level was significant higher than when the BL21(DE3) and BL21(DE3)-pLysS strains were used . Approximately 18% rare E. coli codons are presented in PiCV cap gene, and most of rare codons are basic amino acid residues, such as lysine (K) and arginine (R) and are presented near the 5’-end of the cap gene. Using GeneOptimizer software, the codons of the cap gene were optimized without altering the amino acid sequence of the protein to give a gene sequence that had the preferred codon usage of E. coli. As illustrated in Figure opt. Three recombinant constructs were then created using the codon optimized cap gene sequence and then transformed into E. coli to explore expression levels  or E. coli BL21(DE3)-pLysS were used as host to express the His-rCapopt or the GST-rCap protein, the expression proteins were almost undetectable after IPTG induction  and BL21(DE3)-pLysS strains harbored the pGST-rCapopt plasmid, rCapopt protein was produced at significant levels after IPTG induction -pLysS strain was 394.2 ± 26.1 µg/ml, which is higher than that of the GST-rCap protein at 119.2 ± 17.0 µg/ml when the latter protein is expressed in the BL21(DE3)-RIPL strain -pLysS was even higher than that of BL21(DE3) -pLysS was 1.3 fold higher than that of the GST-rCapopt protein expressed in the BL21(DE3) strain  and BL21(DE3)-pLysS strains also produced a very similar pattern to that of the different E. coli strains expressing GST-rCapopt protein -pLysS strain after IPTG induction for 4 h reached 544.5 ± 33.2 µg/ml at the same optical density (OD) as the cultures, which represents a 5.07 fold increase over Trx-His-rCap in BL21(DE3)-pLysS by densitometric analysis -pLysS was higher than that of Trx-His-rCapopt protein at 283.3 ± 9.0 µg/ml using BL21(DE3) strain and IPTG induction  or in BL21(DE3)pLysS with either the GST or the Trx-His fusion tag.Furthermore, the quantitative yield for the Trx-His-rCaphe BL21DE-pLysS sthe BL21DE-pLysS stopt protein, the solubility of expressed two rCapopt fusion proteins, GST-rCapopt and Trx-His-rCapopt was explored. E. coli BL21(DE3)pLysS cells over-expressing either GST-rCapopt or Trx-His-rCapopt was separated into the supernatant and pellet fractions after sonication of a suspension of harvested cells. The analysis by SDS-PAGE demonstrated that both GST-rCapopt and Trx-His-rCapopt protein existed in soluble and insoluble forms pLysS cells were used. Therefore, purification of the E. coli-expressed GST-rCapopt protein was then carried using a GST affinity column. After affinity chromatography combined with on-column cleavage by thrombin, the presence of collected soluble rCap protein was clearly detectable by SDS-PAGE analysis . At present, no vaccine is available to prevent PiCV infection. Among circovirus, capsid proteins have been investigated as to their usefulness as immunogens for developing subunit vaccines -15. The E. coli. HoweverE. coli remains the most attractive expression system when assessing the expression of a heterologous protein for many different purposes [E. coli expression system to express a heterologous recombinant protein has several advantages; these include cost-effectiveness, ease of production, time-saving and others. In this study, we have successfully produced for the first time the full-length Cap protein of PiCV using an E. coli expression system Previously, it has been suggested that the Cap protein is likely to be the sole structural protein of PiCV and this protein thus controls viral capsid assembly. Thus, have purified full-length Cap protein will help with the detailed study of PiCV's structure biology and it will also help with PiCV vaccine development and the production of a serodiagnostic kit for PiCV detection. The PiCV Cap protein has been demonstrated to have antigenic activity and to be able to recognize by PiCV specific antibodies [E. coli approach to producing PiCV Cap protein has been poor protein expression and low protein solubility. Previous studies have shown that the production of recombinant Cap protein using an E. coli expression is relatively difficult and therefore this has become a bottle-neck [purposes . Using atibodies ,12. Howetle-neck . This stE. coli. These factors include cultivation parameters, the fusing of an affinity tag of one type or another to the target protein and the optimization for E. coli of the codon usage of the foreign gene. [et al. described how the expression of the Cap protein of porcine circovirus (PCV) was successfully improved in E. coli by fusing the maltose-binding protein (MBP) to the target protein [E. coli significantly compared to a His × 6 tag [E. coli expressed proteins more than other tags; this occurs perhaps by promoting the correct folding of their selected fusion partner [A number of different strategies are available when improving protein expression and enhancing the protein solubility in gn gene. ,17,18. T protein , but the protein . Similar × 6 tag . Thus so partner ,19.http://www.imtech.res.in/raghava/dnabinder/submit.html). In previous studies, this N-terminal regions of capsid proteins have often caused problems with recombinant protein expression using a prokaryotic system [E. coli; nonetheless, the usefulness of the truncated protein for diagnosis or for the development of a subunit vaccine is likely to be hampered. Obviously only the intact capsid protein contains all of the epitopes for elicitation of virus neutralizing antibodies by the host. Thus, it is best to express full-length PiCV capsid protein rather than a truncated form when developing a vaccine or a diagnostic kit.Like porcine circovirus (PCV), beak and feather disease virus (BFDV) and chicken anemia virus (CAV), PiCV is also rich in basic amino acid residues close to the N-terminus of the capsid protein. This region has been predicted to be a nuclear localizing sequence and a nucleic acid binding domain via the DNAbinder software package -RIPL, when used to express Trx-His-rCap, gave the highest level of protein expression, significantly higher than any other combination  and BL21(DE3)pLysS, was able to improve protein production and yield. With both GST-Capopt and Trx-His-Capopt, expression was better with BL21(DE3) and produced more Cap protein than BL21(DE3)-RIPL pLysS. However, these effect were not present during the production of GST-Capopt protein using E. coli BL21(DE3)pLysS at high expression levels. The superiority of Trx-His-Capopt expression in BL21(DE3)pLysS may be due to the presence in the strain of the pLysS plasmid during protein induction. The cytotoxicity tolerance of BL21(DE3)pLysS might be associated with the expression of T7 lysozyme which attenuates transcription leakage by T7 RNA polymerase. However, this phenomenon was not significant when GST-Capopt was expressed grown using BL21(DE3)pLysS. GST-Capopt was decreased when there was induction by IPTG. One possibility is that the cytotoxicity of GST-Capopt may be higher that that of Trx-His-Capopt in BL21(DE3)pLysS. In other words, a “protein burden” may not yet have been encountered when BL21(DE3)pLysS was used to express Trx-His-Capopt. Overall, we concluded that BL21(DE3)pLysS is the preferred choice for expressing Trx-His-Capopt.We also investigated which of two different recombinant s, BL21DE and BL21s, BL21DE and BL21opt protein is superior to that of Trx-His-Capopt. Furthermore, it is very easy to apply the protein to a GST-affinity column in order to carry out a protein purification. Moreover, the Cap protein contains basic amino acid residues rich at its N-terminus. Such highly positive charge amino acids within the recombinant Cap protein allow easy polishing by cation exchange column as part of down-stream processing. Therefore, it seems likely that, a higher purity of Cap protein can be obtained when an affinity column is combined with an ion-exchange chromatography during vaccine development.The solubility of recombinant Cap protein is a potential problem and in this context the solubility of GST-CapE. coli-expressed recombinant PiCV Cap protein. All tested PiCV-infected pigeon sera were able to demonstrate that E. coli-expressed recombinant PiCV Cap protein has the correct antigenic activities. This might be a result of the E. coli-expressed recombinant Cap protein displaying all of the appropriate protein antigenic regions on the protein surface for recognition by the pigeons' antibodies. However, rCap protein when used against some pigeon sera did not show very strong antigenicity. This perhaps suggests that the titers of antibodies against PiCV Cap protein have various levels in pigeons and such variation might explain in lower performance of antigenicity in certain birds.In this study, positive PiCV-infected pigeon sera were used to evaluate the antigenic activity of the E. coli and by optimizing the codon usage of the polypeptide, it was possible to increase the yield of Cap fusion protein significantly. In this context, a convenient and cost-effective strategy for increasing the expression of Cap protein, which was used herein, was the direct engineering of the codons of the Cap protein to fit the E. coli codon preferences. This approach paves the way for the large-scale efficient production of the PiCV Cap protein. In the future, this will also allow recombinant Cap protein to be used as a potential antigen for the development of a PiCV diagnostic test.In conclusion, using a prokaryotic system, the optimal expression of recombinant full-length PiCV Cap protein was established successfully during this study. Furthermore, by fusing the Cap protein to an affinity tag, by using the appropriate preferred E. coli strains, BL21(DE3) , BL21(DE3)CodonPlus-RIPL  and BL21(DE3)pLysS  were used and maintained at 37°C in the Luria-Bertani (LB) medium . First, 0.5 mL of an overnight culture was inoculated into 50 mL LB medium to allow strain activation by growth at 37°C for around 3 hours, by which time the optical density of culture had reach 0.5 of OD600. These bacterial cells were then used for transformation.Three commercial cap gene that encodes the full-length PiCV capsid protein was synthesized by Genemark Biosci & Tech Co.  based on the published sequence . This cDNA was cloned into either pET28a, pET32a  or pGEX-4T-1  individually using EcoR1 and XhoI  restriction sites. The resulting recombinant plasmids were designated pHis-Cap, pTrx-His-Cap and pGST-Cap, respectively  chemically competent E. coli for maintenance of the recombinant plasmids. Transformants that containing a insert of the correct size were then confirmed as correct by restriction enzyme digestion and by DNA sequence analysis.A 822 bp of cDNA fragment consisting of the opt protein, all the constructed recombinant plasmids carrying either the cap gene or the codon-optimized cap gene, as described in Figure E. coli strains to allow evaluation of protein expression. Three commercial E. coli host strains, BL21(DE3), BL21(DE3)CodonPlus-RIPL and BL21 (DE3)pLysS, each with a different recombinant construction, were used for protein induction and expression. The culture conditions, the composition of the LB medium and the protein induction condition of these recombinant strains have been described previously [opt proteins were analyzed by 12.5% SDS-PAGE and Western-blotting using a monoclonal anti-His antibody  or a monoclonal anti-GST antibody .To express the recombinant rCap or rCapeviously . After Ieviously . Samplesopt protein was purified from cells expressing the GST-rCapopt protein. This was carried out by spinning down 50 mL of culture supernatant and resuspended the pellet in GST resin binding buffer . The mixture was then sonicated on ice three times for 3 minutes using a 20% pulsed activity cycle (MISONIX Sonicator® 300). Next, the lysate was centrifuged for 10 min at 10,000 rpm to remove the cell debris. The resulting cell supernatant was loaded onto a GSTrap FF affinity column  for protein purification using the standard procedure described in a previous study [Recombinant rCapus study . The totopt protein, E. coli expressed GST-rCapopt protein that had been purified by GSTrap FF column was used. The rCapopt protein that had been eluted from the GSTrap FF column was loaded onto a SP cation exchange chromatography column  for further purification. The cation exchange column-purified rCapopt protein was then analyzed by 12.5% SDS-PAGE. The relevant band was then cut out from the 12.5% SDS-PAGE gel after coomassie blue staining and digested with trypsin. The resulting peptides were subjected to the MALDI-TOF-MS mass spectrometry (ESI-QUAD-TOF) to allow amino acid sequence identification of the protein, as described in a previous study [To confirm the identity of the recombinant Capus study .The author declares that they have no competing interests.MSL participated in this study design, performed the experiments and in the writing of the manuscript. GHL performed the experiments, study design and participated in the construction of the plasmids. YYL participated in the experiments on protein antigenicity and MKL, YCL, and YLT participated in the protein purification step and determining protein solubility. JTCT participated in the data analysis and the writing of the manuscript. HJC and WTC coordinated the study and participated in performing ELISA assay. All authors read and approved the final manuscript.


Anxiety vulnerable individuals exhibit enhanced acquisition of conditioned eyeblinks as well as enhanced proactive interference from conditioned stimulus (CS) or unconditioned stimulus (US) alone pre-exposures (Holloway et al.,  Recent work has focused on a learning diathesis model of anxiety disorders involving anxiety vulnerability and associative learning  tone with an unconditional stimulus (US) corneal air puff which results in learning a conditioned response (CR) eyeblink to the previously neutral CS. Partial reinforcement with either CS or US alone trials inter-mixed with CS-US paired trials produced enhanced acquisition of conditioned responses in anxiety vulnerable individuals that was greater than that observed with 100% CS-US paired trials  prior to paired CS-US training results in slower acquisition of CR. Holloway et al.  assessedHowever, some aspects of the analyses reported by Holloway et al.  bring inThe reporting of measures of anxiety vulnerability was also incomplete. Holloway et al.  collecteHolloway et al.  also tesBased on these unanswered questions, we wanted to extend the US pre-exposure effect from the Holloway et al.  study wiWe hypothesized that US alone pre-exposures would disrupt subsequent CR acquisition as compared to context alone pre-exposure. We also hypothesized that while enhanced acquisition would be evident in BI individuals in the context (no stimulus) pre-exposure condition, there would be no BI enhancement effect in the US alone pre-exposure condition. We also hypothesized that there would be no significant differences in spontaneous blinking during context and US alone pre-exposures between anxiety vulnerable and non-vulnerable individuals.Sixty-six college-aged students were recruited from the University of Northern Colorado, School of Psychological Sciences. Students voluntarily participated to receive class credit or extra credit for psychology coursework. Our sample consisted of 45 females and 21 males with a mean age of 22.4 years  and mean education of 13.7 years . Informed consent was obtained in accordance with procedures approved by the University of Northern Colorado Institutional Review Board.The eyeblink conditioning apparatus and procedures were similar to that previously described  and non-inhibited (NI) groups based on a median split of AMBI scores. This methodology was based on previous eyeblink conditioning studies with BI , and that they were to remain awake during the testing session. Participants were then fitted with EMG electrodes and headphones, EMG signal quality was verified, and the conditioning program was started. Participants received either US alone or context alone  pre-exposures for 30 trials, followed immediately by 30 CS-US paired trials. Paired CS-US trials included a 500 ms/1200 Hz pure tone CS overlapping and co-terminating with the 50 ms US air puff. The ITI varied pseudo-randomly between 30 ± 5 s.EMG data were evaluated on a trial-by-trial basis for all participants. Processing of eyeblink responses followed methods previously reported , equipment malfunction, or incomplete session data , were discarded and not used for further analysis. In addition, the data of participants who had excessive levels of spontaneous blinks or alpha responses (80% or more CRs starting in the first block) were excluded from data analysis.p < 0.05.To examine the main effects and interactions of anxiety vulnerability and CR acquisition, the 60 trial conditioning session was divided into 10 trial blocks and evaluated independently for 30 pre-exposure trials (3 blocks) and 30 acquisition trials (3 blocks). Between group measures included pre-exposure , and BI , with Block as a within subject measure. Pairwise comparisons were planned between the high and low BI groups as well as between the two pre-exposure conditions. The level of significance was set at Inspection of the eyeblink data resulted in rejection of data from 28 participants . Most exclusions were due to excessive spontaneous blinking or alpha responses from the start of conditioning. Psychometric and demographic data for the remaining 38 participants including the BI and NI groups for the context and US alone pre-exposure conditions are summarized in Table p = 0.254) or BI (p = 0.46) for eyeblinks recorded during the pre-exposure phase. There was a significant interaction of BI × block for the spontaneous blink rate during context pre-exposure trials such that the high BI group had higher initial blinking than the NI group and that was reduced as context pre-exposure continued  = 3.509, p < 0.05). No other main effects or significant interactions were found in context or US pre-exposure conditions. An example of an unconditioned response (UR) on an air puff alone pre-exposure trial is shown in Figure Eyeblinks were monitored during the 450 ms window prior to the time of the US presentation during which the CS would occur in the CS-US paired trials. No stimuli were presented during this window in either the US alone or context alone pre-exposure trials. There was no main effect of US pre-exposure  = 24.383, p < 0.001) as shown in Figure p = 0.486), block × pre-exposure (p = 0.226) or block × BI × pre-exposure (p = 0.166).All participants acquired CRs. An example of a conditioned response to a paired tone and air puff acquisition trial is shown in Figure F = 14.864, p < 0.001) as shown in Figure Pre-exposure of the US alone produced a proactive interference effect such that individuals pre-exposed to the US alone exhibited fewer CRs than individuals pre-exposed to the context only. This finding was supported by a main effect of pre-exposure  = 5.460, p < 0.05). This interaction is evident in Figure F = 34.134, p < 0.001) as shown in Figure F = 0.581, p = 0.457) as shown in Figure p > 0.90) or BI individuals (p > 0.010).There was also a significant pre-exposure × BI interaction  = 0.001, p = 0.974) as shown in Figure F = 5.460, p < 0.05). This interaction is evident in Figure F = 4.976, p < 0.05) as shown in Figure F = 2.438, p = 0.138) as shown in Figure p = 0.383) and US air puff alone conditions (p = 0.75).There was no overall effect of BI  in human eyeblink conditioning . The neural substrates of LI were first identified as the hippocampus with non-selective lesions  inbred rat strain. WKY rats are a BI strain that has been suggested to be a model of stress and anxiety vulnerability. WKYs emit more CRs and achieve higher asymptotic performance during classical eyeblink conditioning than the outbred Sprague Dawley (SD) strain (Ricart et al., The major finding of the current work is that anxiety vulnerable individuals appear more sensitive to pre-exposure effects like proactive interference than non-vulnerable individuals. This proactive interference is so strong in BI individuals as to disrupt the enhanced acquisition of conditioned eyeblink observed with BI individuals pre-exposed to the context alone. Future work should test whether PTSD sufferers exhibit similar effects with US alone pre-exposures and proactive interference.PTSD has been hypothesized to occur through enhanced associative learning Pitman, . Prior wMTA and DM were involved in the design of the study. MTA collected the data and prepared the manuscript. MTA and DM were involved in the revisions to the manuscript.Funding was provided by the University of Northern Colorado and the Stress and Motivated Behavior Institute.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Platelets are frequently altered in hepatocellular carcinoma (HCC) patients. Platelet lysates (hPL) can enhance HCC cell growth and decrease apoptosis. The aims were to evaluate whether hPL can modulate the actions of Sorafenib or Regorafenib, two clinical HCC multikinase antagonists.Several human HCC cell lines were grown in the presence and absence of Sorafenib or Regorafenib, with or without hPL. Growth was measured by MTT assay, apoptosis was assessed by Annexin V and by western blot, and autophagy and MAPK growth signaling were also measured by western blot, and migration and invasion were measured by standard in vitro assays.Both Sorafenib and Regorafenib-mediated inhibition of cell growth, migration and invasion were all antagonized by hPL. Drug-mediated apoptosis and decrease in phospho-ERK levels were both blocked by hPL, which also increased anti-apoptotic phospho-STAT, Bax and Bcl-xL levels. Preliminary data, obtained with epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I), included in hPL, revealed that these factors were able to antagonized Sorafenib in a proliferation assay, in particular when used in combination.Platelet factors can antagonize Sorafenib or Regorafenib-mediated growth inhibition and apoptosis in HCC cells. The modulation of platelet activity or numbers has the potential to enhance multikinase drug actions. Platelet activity has been known for a long time to be altered in the presence of cancer, with venous thrombosis being recognized in association with occult malignancy,2. In adPLC/PRF/5, Hep3B and HepG2 cells were obtained from the ATCC and were cultured as previously described.Recombinant human EGF was purchased from PeproTech , mouse recombinant IGF-I from Calbiochem  and serotonin from Sigma-Aldrich , all the growth factors were dissolved in water.The platelet samples were collected from healthy volunteers. The study protocol was approved by the institutional review boards of the University of Bari and “Saverio de Bellis” Institute of Castellana G. (BA), Italy. Additionally, written informed consent was obtained from participants for the use of their blood in this study.The platelet-rich plasma was obtained using an automated hemapheresis procedure in a local blood transfusion center. The platelets obtained from different volunteers were mixed and then divided into aliquots. Each aliquot was subjected to several freeze-thaw cycles to disrupt their membranes and release the growth factors stored in the granules .7) or equivalent percentage of FBS in presence of 1 μM (HepG2 cell line) or 2.5 μM (Hep3B and PLC/RFP/5) of Sorafenib or Regorafenib. In the same growth condition HCC cell lines were cultured in presence of EGF 10, 25 mg/ml, IGF-I 50, 100 mg/ml and serotonin 1, 10 μM with or without Sorafenib 1 μM.Proliferation assay was performed as recently described,20. The Medium AFP levels were measured using an automated system  by a chemoluminescent immunometric method. Sample measurements over the calibration range were automatically re-analyzed according to manufacture’s instructions.A scratch assay was performed as previously described,20. BrieCell invasion assays were performed using Matrigel -coated Transwells  as previously described (19). Briefly, 2.5 μM Sorafenib or Regorafenib treated cells were suspended in low serum medium. Medium containing different hPL or FBS concentrations was added to the bottom wells. After incubation of 24 h, the invading cells were fixed and stained. The images were acquired and analyzed counting the cells with Image J Software . Values obtained were expressed as fold increase of invading cells, setting the cell counts of control cells as one. Results were representative of three independent experiments.7 platelets/ml or with an equivalent percentage of serum (control cells) for 48 h. The cells were then processed as described in the user’s guide.The Muse Annexin V/Dead Cell Assay Kit  for quantitative analysis of live, early/late apoptotic and dead cells was used with a Muse Cell Analyzer (Millipore). Briefly, the assay utilizes Annexin V to detect PS on the external membrane of apoptotic cells. A dead cell marker (7-AAD) is also used. PLC/PRF/5 cell line, including positive and negative controls, were cultured in 1% FBS medium supplemented with a volume of hPL corresponding to 3.75 × 10The Muse Caspase-3/7 kit (Millipore) permits simultaneous evaluation of apoptotic status based on Caspase-3 and -7 activation and cellular plasma membrane permeabilization (cell death). The assay provides relative percentage of cells that are live, early/late apoptotic or dead. Cells were cultured as described above and processed according to the user’s guide.We analyzed the MAPK signaling and anti-apoptosis markers in Hep3B cells treated with 2.5 μM Sorafenib or Regorafenib and hPL by Western blot, as previously described,20. In b705, Ser727) (P-STAT3), AKT and phospho-AKT (P-AKT), survivin, Bcl-xL, Bax, Bim and β-actin . After three washes, incubation was followed by the reaction with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The immunoreactive bands were visualized and analyzed using the enhanced chemiluminescence detection reagents , according to the manufacturer’s instructions, and chemiluminescence detection system .The primary antibodies were directed against the following proteins: ERK and phospho-ERK (P-ERK), JNK and phospho-JNK (P-JNK), p38 and phospho-p38 (P-p38), STAT3 and phospho-STAT3  was used for all statistical analysis. Mann–Whitney nonparametric test was employed to assess the statistical significance of differences between two groups. For multiple comparisons was used one-way Anova test followed by appropriate post-test. P-values of <0.05 were considered statistically significant. All experiments were done in triplicate and data are presented as mean ± standard deviation (SD).hPL were previously examined for the ability to stimulate human HCC cell line growth. Hep3B, 7 platelets in all the HCC cell lines, compared with treatments with Regorafenib or Sorafenib in presence of FBS. Figure 7 platelets.Cells were also treated in the absence or presence of increasing concentrations of hPL. A significant increase of cell growth was detected in presence of hPL from 3.75 × 10Comparing the growth in these different conditions by MTT assay, it was clear that increasing the serum concentration more than 1% had not significant influence on PLTs antagonism , an HCC cell growth marker, were also measured. We found that Sorafenib-mediated inhibition of AFP levels was also antagonized by the presence of hPL Figure H.7 platelets. We found that hPL caused an increase in P-ERK levels, as well as for P-p38 and P-STAT3 (Ser and Tyr). By contrast, P-JNK levels were not modified by the presence or absence of hPL , IGF  and EGF  alone and in combination. The effect on proliferation, evaluated by MTT assay after 48 h, was significant only with EGF, while serotonin and IGF were effective only when used in combination. Figure We report here for the first time, the antagonizing effects of platelet extracts on growth inhibition in several HCC cell lines, that was mediated by Sorafenib or Regorafenib. Both agents were similarly antagonized by hPL. Furthermore, the previously demonstrated inhibition of AFP secretion by these drugs, was also antagonized. A main consequence of each drug is a decrease in phospho-ERK levels, secondary to Raf inhibition. hPL antagonized this early consequence of the drug action, without change in ERK levels. There was also an early and strong antagonism of the previously noted inhibitory effects of drug on phospho-p38 levels, and simConsistent with our findings of increased phospho-STAT3 levels, we also found an increase in the levels of anti-apoptotic Bcl-xL and survivin and a decrease in the levels of pro-apoptotic Bim and Bax, consequent to hPL action.Due to the important role of platelets in the metastasis mechanisms of many tumors,21, we ePlatelets are the source of multiple growth factors, cytokines and inflammatory mediators.Included among them are EGF, IGF-I, fibroblast growth factor (FGF), platelet derived growth factor (PDGF) and serotonin, the modulation of each having been shown to alter cancer chemotherapy sensitivity or resistance-30. PrelInterestingly, the clinical insulin modulator and diabetes drug, metformin and the The recent finding that platelet inhibitors reduce hepatitis B associated experimental HCC has led The current results give support to the idea that platelet inhibitors might also be useful in the drug therapy of patients with unresectable HCC, provided their platelet levels and coagulation systems are normal.HCC: Hepatocellular carcinoma; hPL: Human Platelets lysate; PVT: Portal vein thrombosis; ERK: Extracellular signal-regulated kinase; JNK: c-Jun NH2-terminal kinase; STAT: Signal transducer and activator of transcription-3; WB: Western blot; MTT: 3--2,5-diphenyltetrazolium bromide; BrdU: 5-bromo-2′-deoxy-uridine; AFP: Alpha-fetoprotein; EGF: Epidermal growth factor; IGF-I: Insulin-like growth factor-I.The authors declare that they have no competing interests.BIC involved in interpretation of clinical data, conception and design of the translational research study in vitro and wrote the first draft of the manuscript; RD, MGR and CL participated equally at the design, execution and interpretation of the experiments; NC performed Western blot experiments; GG participated in blood collection, isolation and count of platelets; CM and AC provided overall supervision for conducting the study and involved in manuscript revision and presentation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/351/prepub